Internally quenched fluorescent peptides provide insights into underexplored and reversible post-translational modifications

Internally quenched fluorescent (IQF) peptides offer a powerful, modular platform for studying the enzymatic dynamics of post-translational modifications (PTMs) on lysine and arginine. Here we report a versatile IQF system that enables monitoring of PTM installation and removal <i>via</i> proteolytic cleavage by trypsin. This platform is compatible with both native PTMs and PTM mimetics, including acetylation, various other acylations, mono-/di-/trimethylation and citrullination across both histone and non-histone derived peptide substrates. Using synthetically accessible thialysine and thiaarginine analogs, we developed cysteine conjugation chemistries to access a wide array of PTM mimics, including novel reagents for lysine lactylation, β-hydroxybutyrylation and methyl-acetylation. Application of the system revealed distinct substrate preferences and site-specific activities for enzymes such as SIRT3, HDAC2, HDAC6, KDM3A, KDM4A and PAD4. Notably, the system uncovered enzymatic selectivity for acyl chain type and methylation state and demonstrated resistance of the emerging PTM methyl-acetyllysine to erasers. The system was also used to study the recently reported reversibility of acylation modifications by HDAC2 and 6 and is capable of evaluating enzymatic crosstalk between neighboring post-translational modifications. Our platform's adaptability and readout simplicity offer a generalizable chemical biology toolkit for PTM profiling, enzyme characterization, and inhibitor discovery.